Hantupeptin A, a cytotoxic cyclic depsipeptide from a Singapore collection of Lyngbya majuscula

Article abstract of DOI:10.1021/np800448t

Chemical investigation of the marine cyanobacterium Lyngbya majuscula from Pulau Hantu Besar, Singapore, has led to the isolation of a cyclodepsipeptide, hantupeptin A (1). The planar structure of 1 was assigned on the basis of extensive 1D and 2D NMR spe

Full text of DOI:10.1021/np800448t

J. Nat. Prod. 2009, 72, 29–32
29
Hantupeptin A, a Cytotoxic Cyclic Depsipeptide from a Singapore Collection of
Lyngbya majuscula
Ashootosh Tripathi,† Jonathan Puddick,‡ Michele R. Prinsep,‡ Peter Peng Foo Lee,† and Lik Tong Tan*,†
Natural Sciences and Science Education, National Institute of Education, Nanyang Technological UniVersity, 1 Nanyang Walk,
Singapore 637616, Singapore, and Department of Chemistry, UniVersity of Waikato, PriVate Bag 3105, Hamilton 3205, New Zealand
ReceiVed July 20, 2008
Chemical investigation of the marine cyanobacterium Lyngbya majuscula from Pulau Hantu Besar, Singapore, has led
to the isolation of a cyclodepsipeptide, hantupeptin A (1). The planar structure of 1 was assigned on the basis of extensive
1D and 2D NMR spectroscopic experiments. The absolute configuration of the amino and hydroxyl acid residues in the
molecule was determined by application of the advanced Marfey method, chiral HPLC analysis, and Mosher’s method.
Hantupeptin A showed cytotoxicity to MOLT-4 leukemia cells and MCF-7 breast cancer cells with IC50 values of 32
and 4.0 µM, respectively.
Filamentous marine cyanobacteria are an established source for
a prodigious array of unique bioactive secondary metabolites.1,2
These metabolites exhibit a variety of biological activities including
immunosuppressant, antiproliferative, and antimicrobial activities.4,5
Interestingly, approximately 40% of the cyanobacterial compounds
possess anticancer/antitumor activity, making them invaluable as
potential therapeutic leads.6 Recent examples of potent marine
cyanobacterial metabolites include coibamide A,7a largazole,7b and
somocystinamide A.7c The benthic filamentous cyanobacterium
Lyngbya majuscula, in particular, has proven to be an exceptional
source of novel potential pharmaceuticals.2 Among the diverse
classes of compounds being discovered from this species, a
substantial amount belongs to either the polypeptide or hybrid
polyketide-polypeptide structural classes.1-3 In spite of the
polarity. The fraction eluted with 80% EtOAc/20% hexane was
subjected to SEP PAK C18 solid-phase fractionation followed by
reversed-phase HPLC to yield hantupeptin A (1).
chemical richness of these marine microorganisms, no studies have
been conducted on the natural products chemistry of marine
cyanobacteria from Singapore.
Hantupeptin A (1) was isolated as an amorphous solid, and
HRESIMS of the molecule provided a molecular formula of
C41H60N4O8 (14 degrees of unsaturation) based on the [M + H]+
and pseudomolecular ion [M + Na]+ peaks at m/z 737.4484 and
759.4310, respectively. The molecule seems to adopt at least two
conformers as observed in the 1D NMR data when measured in
CDCl3. Chemical signals due to the minor conformer were
diminished when 1H NMR data were acquired at higher temperature
(at 50 °C) during the variable-temperature experiments (see below).
The presence of a peptidic molecule was evident from the 1H NMR
spectrum, which showed two tertiary amide N-Me 3H singlets at δ
3.07 and 2.60 as well as a characteristic secondary amide NH
resonance occurring as a doublet at δ 6.31. Furthermore, the 13C
NMR spectrum of 1 exhibited the presence of six carbonyl carbons
attributable to ester/amide functionalities and a monosubstituted
phenyl ring (δ 136.6, 129.9, 128.8, and 127.1) system (Table 1).
These functionalities accounted for 10 of the 14 degrees of
unsaturation.
In our ongoing efforts toward finding novel and pharmacologi-
cally active marine cyanobacterial metabolites, a detailed biological
investigation of L. majuscula sampled from intertidal regions at
different islands off the southern coast of Singapore was undertaken.
Preliminary data from the brine shrimp toxicity and MTT cyto-
toxicity (based on the MOLT-4 cell line) assays revealed a high
incidence of biological activities in these cyanobacterial organic
extracts (unpublished data). Furthermore, L. majuscula procured
from the western lagoon of Pulau Hantu Besar, Singapore, displayed
the most significant activities. Chemical investigation of this strain
resulted in the isolation of two new fatty acid amides, besarhana-
mides A and B, recently reported in the literature.8 Further
examination of other active fractions has now led to the isolation
of a new cyclic depsipeptide. Herein, we wish to report a bioassay-
guided isolation and structure elucidation of hantupeptin A (1),
which was also shown to possess high levels of cytotoxicity toward
cancer cells.
Extensive NMR analyses based on a combination of 1D and 2D
NMR data of 1 allowed construction of six partial structures. Four
standard amino acid residues were deduced as N-methylisoleucine
(N-Me-Ile), N-methylvaline (N-Me-Val), valine (Val), and proline
Results and Discussion
Samples of the marine cyanobacterium Lyngbya majuscula were
collected by hand during low tides from the western lagoon of Pulau
Hantu Besar, Singapore, and stored in 70% ethanol. The organic
extract was prepared using CHCl3/MeOH (1:1) and subsequently
fractionated by vacuum flash chromatography (VFC) based on a
combination of hexanes, EtOAc, and MeOH with increasing
1
(Pro) (Figure 1). A fifth residue exhibited H NMR resonances at
δ 7.21-7.53 (H-25 to H-29), 2.94 (H-23a), 3.18 (H-23b), and 5.51
(H-22), which are characteristic of a phenylalanine residue.
However, the HSQC spectrum showed that H-22 was attached to
an oxymethine carbon (δ 73.2) and thus was more consistent with
a 3-phenyllactic acid (Pla) moiety (Figure 1). The sixth and final
residue required a composition of C9H12O2 to complete the
molecular formula of 1. From TOCSY, HMBC, and HSQC data,
* Corresponding author. Tel: +65 6790 3820. Fax: +65 6896 9432.
E-mail: liktong.tan@nie.edu.sg.
† Nanyang Technological University.
‡ University of Waikato.
10.1021/np800448t CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/18/2008

30 Journal of Natural Products, 2009, Vol. 72, No. 1
Tripathi et al.
Table 1. 1D NMR Data for the Major Conformer of Hantupeptin A (1) in CDCl3
unit
C/H no.
δH (J in Hz)
δC
COSY
H-3, H-9
H-4a, H-4b, H-2
H-3, H-4b, H-5
H-4a
HMBC
Hmoya
1
2
3
4a
4b
5
6a
171.1, qC
42.6, CH
77.5, CH
25.6, CH2
3.31, m (6.5, 2.8)
4.91, dt (10.6, 2.8)
1.67, m
2.13, m
1.68, m
1, 3, 9
3
25.4, CH2
18.4, CH2
H-4b, H-6a, H-6b
H-5, H-6b
2.24, m
5, 7, 8
6b
7
8
1.50, m
83.9, qC
69.1, CH
20.1, CH3
165.8, qC
66.2, CH
27.8, CH
14.5, CH3
11.5, CH3
29.2, CH3
173.8, qC
57.9, CH
29.5, CH2
1.98, t
1.31, d
6, 7
9
H-2
1, 2, 3
N-Me-Val
10
11
12
13
14
15
16
17
18a
18b
19a
19b
20
21
22
23a
23b
24
25/29
26/28
27
30
31
32
33a
33b
34
35
36
37
38
39
40
41
NH
4.07, d (10.2)
2.16, m
0.92
0.92
2.60, s
H-12
10
H-13, H-14
H-12
12
H-12
12
11, 16
Pro
4.98, dd (8.4, 4.7)
2.29, m
1.85, m
2.00, m
2.09, m
H-18a, H-18b
H-19b, H-18b
H-18a
19
27.9, CH2
H-20, H-19b
H-19a, H-18b
H-19a, H-19b
3.60, m
47.4, CH2
171.4, qC
73.2, CH
37.4, CH2
18, 19
Pla
5.51, dd (9.8, 4.1)
2.94, dd (14.8, 9.8)
3.18, dd (14.8, 4.1)
H-23a, H-23b
H-22, H-23b
H-22, H-23a
23
22, 24, 25/29
22, 25/29, 21
136.6, qC
129.9, CH
128.8, CH
127.1, CH
169.3, qC
64.5, CH
35.0, CH
26.1, CH2
7.53, m
7.28, m
7.21, m
H-26, H-28
H-25, H-29, H-27
H-26, H-28
24, 26, 28
25, 27, 29
26, 27, 28
N-Me-Ile
4.05, d (11.0)
2.13, m
2.23, m
1.51, m
0.90
H-32
30, 32, 35
35
H-31, H-35
H-33b, H-34
H-32, H-33a, H-34
H-33a, H-33b
H-32
32, 34
32, 33
32
16.6, CH3
18.0, CH3
29.5, CH3
172.8, qC
53.2, CH
32.7, CH
20.5, CH3
20.1, CH3
0.92
3.07, s
31, 37
Val
4.78, dd (8.8, 6.4)
2.02, m
0.97
0.97
6.31, d (8.8)
H-39, NH
H-38, H-40, H-41
H-39
H-39
H-38
37, 39
37, 38, 40, 41
38, 39
38, 39
1
the remaining spin system was shown to possess a methine group
at δ 3.31 (H-2)/42.6 (C-2) connected to a methyl group at δ 1.31
(H-9)/20.1 (C-9) as well as to an oxygen-bearing methine at δ 4.91
(H-3)/77.5 (C-3), which was bonded to a methylene group at C-4.
From COSY and TOCSY data, C-4 was shown to be further
extended by two methylene carbons at C-5 and C-6. In addition,
HMBC experiments confirmed the presence of a terminal acetylenic
group with correlations from δ 2.24 (H-6) to carbons at δ 69.1
(C-8) and 83.9 (C-7). These data led to the identification of this
spin system as a 3-hydroxy-2-methyloctynoic acid (Hmoya) residue
(Figure 1). Since Hmoya and Pro accounted for three of four
remaining degrees of unsaturation and with only a single degree
of unsaturation remaining, compound 1 must be a cyclic depsipep-
tide. In addition, HMBC correlations were used to unambiguously
connect these residues, completing its planar structure as shown in
1. Furthermore, the MS/MS spectrum of the molecule provided m/z
peaks that corresponded with fragments obtained from 1, such as
m/z 228 (Val/N-Me-Ile), 246 (Pla/Pro), and 611 (Pla/Pro/N-Me-
Val/Hmoya/Val).
The absolute configuration of all regular and N-Me-Val units in
1 was established as L on the basis of the advanced Marfey
method.9-11 An L-configuration was determined for the Pla moiety
using chiral HPLC analysis. Although, Marfey’s analysis clearly
suggested the L-configuration of the N-Me-Ile moiety, we were
unable to distinguish N-Me-L-allo-Ile and N-Me-L-Ile by this method
due to the lack of standard N-Me-L-allo-Ile. In order to confirm
the L-configuration of the N-Me-Ile in 1, racemization of the N-Me-
L-Ile standard was carried out, and the reaction mixture, containing
both N-Me-L-Ile and N-Me-D-allo-Ile, was subjected to the advanced
Figure 1. Subunits of hantupeptin A (1) with COSY (bold bonds)
and selected HMBC correlations (arrow).

Depsipeptide from a Singapore Lyngbya majuscula
Journal of Natural Products, 2009, Vol. 72, No. 1 31
In comparison, a derivative of hantupeptin A, trungapeptin A, was
reported to be inactive when tested at 10 µg/mL against KB or
LoVo cells.15
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Bellingham Stanley ADP 440 polarimeter. UV and IR spectra
were measured on a Varian Cary 50 UV visible spectrophotometer and
a PerkinElmer spectrum 100 FT-IR spectrophotometer, respectively.
Proton, 13C, and 2D NMR spectra were recorded in CDCl3 on a 400
MHz Bruker NMR spectrometer using the residual solvent signal (δH
at 7.26 and δC at 77.0) as internal standards. Both HRESIMS and
LRMS/MS data were obtained using a Microtof series 89 MALDI TOF
mass spectrometer equipped with an ESI multimode ion source detector.
HPLC isolation of hantupeptin A (1) was conducted on a Shimadzu
LC-8A preparative LC and Shimadzu SPD-M10A VP diode array
detector, while an Agilent 1100 series coupled with an Agilent LC/
MSD trap XCT mass spectrometer equipped with an ESI interface
system was used for the detection of the Marfey-derivatized L/D-valine,
proline, N-methylvaline moiety from hantupeptin A. Chiral analysis
was carried out using a Waters 2790 Alliance HPLC (Milford, MA)
equipped with a photodiode array detector. Cell viability in 96-well
plates was measured using a Bio Rad Benchmark plus microplate reader.
Biological Material. About 1.0 L of the filamentous benthic marine
cyanobacterium Lyngbya majuscula was collected by hand from the
western lagoon of Pulau Hantu Besar (1°13′31.44′′ N, 103°44′55.89′′
XXXX) during low tides on June 29, 2005, and stored in 70% aqueous
EtOH at -20 °C before extraction. A voucher specimen of this
microalga is maintained at the National Institute of Education,
Singapore, under the code TLT/PHB/002.
Figure 2. Preparation of 3a and 3b and ∆δ values (×10-3 ppm).
Isolation and Purification. Extraction of Lyngbya majuscula was
carried out repeatedly using CHCl3/MeOH (1:1) to produce about 1.0 g
of crude organic extract. The extract was then subjected to vacuum
flash chromatography (VFC) on normal-phase Si using a combination
of hexanes, EtOAc, and MeOH of increasing polarity. Nine fractions
were obtained, and solvents were removed in Vacuo using a rotary
evaporator before storage in 4 dram vials in CHCl3. All fractions were
assayed at 100 and 10 ppm in the brine shrimp (Artemia salina) toxicity
assay.
Marfey analysis using LC-MS. Both the retention times of the
L-Marfey-derivatized N-Me-L-Ile standard and the N-Me-Ile unit
in the hydrolyzate of 1 were similar, and this was confirmed by
coelution.
The absolute stereochemistry at C-3 was determined using
Mosher’s analysis12 following methanolysis of 1 and isolation of
fragment 2 (Figure 2), the structure of which was confirmed by 1H
NMR data. Subsequently, treatment of 2 with Mosher’s reagents
showed unambiguously that C-3 had the S-configuration (for ∆δ
values, see Figure 2). The Hmoya unit has also been reported in a
number of marine-derived compounds such as onchidin B,
kulomo’opunalide 1, kulomo’opunalide 2, and trungapeptin A,
having 1H and 13C NMR signals well within the comparable limits
of 1.13-15
Variable-temperature NMR experiments were performed on
hantupeptin A (1) in order to probe the degree and nature of the
amide proton (Val) participating in intramolecular hydrogen bonding
within the molecule.16 Proton NMR spectra of 1 were acquired
over a range of temperatures in CDCl3, and the respective ∆δ/∆T
(in ppb/°C) value of the amide proton was calculated as -0.1. This
value is small enough to suggest that the NH of Val is involved in
an intramolecular H-bond.
The solution confirmation of hantupeptin A (1) was predicted
in CDCl3 on the basis of several 2D ROESY experiments using
different mixing times. The observed ROE correlations for 1
included Val-NH (i position) to Hmoya-Hꢀ1 (i+1), Val-NH (i
position) to Hmoya-HR (i+2), Val-NH (i position) to N-Me-Val-
HR (i+3), and Val-HR (i-1) to N-Me-Val-HN-Me (i+4), suggesting
a single turn within the backbone due to the intramolecular
H-bonding between the amide proton of Val and the carbonyl
oxygen atom of N-Me-Val.
Hantupeptin A (1) showed 100% brine shrimp toxicity at 100
and 10 ppm, which was significantly higher than the activity
reported for its close analogue trungapeptin A, which exhibited mild
brine shrimp toxicity.14 Furthermore, in Vitro cytotoxicity testing
of 1 against the leukemia cell line MOLT-4 exhibited an IC50 value
of 32 nM. Compound 1 also displayed significant cytotoxicity in
the breast cancer cell line MCF-7, with an IC50 value of 4.0 µM.
Fraction 7 (100% toxicity at 10 ppm in the brine shrimp toxicity
assay) obtained from VFC of the organic extract of L. majuscula was
subjected to further fractionation on a SEP-PAK RP-18 cartridge using
a combination of MeOH and H2O into four subfractions. The brine
shrimp active subfraction eluted with 10% H2O in MeOH and was
subjected to purification by preparative HPLC [Phenomenex Sphere-
clone 5 µm ODS, 250 × 10.00 mm, 8:2 MeOH/H2O in 50 min at 4.0
mL/min, detected at 215 nm) to elute hantupeptin A (1, 56.2 mg, tR )
15.4 min), yielding approximately 0.5% of crude extract.
Hantupeptin A (1): white, amorphous powder; [R]25D -41.5 (c 1.0,
MeOH); UV (MeOH) λmax 204 nm (log ε 4.4); IR (neat) 3584, 2963,
1736, 1649, 1531, 1452, 1405, 1242, 1184, 1123 cm-1 1H NMR
;
(400.13 MHz, CDCl3) and 13C NMR (100.62 MHz, CDCl3) data, see
Table 1; HRESIMS m/z [M + H]+ 737.4484 (calcd for C41H61N4O8,
737.4489), [M + Na]+ 759.4310 (calcd for C41H60N4O8Na, 759.4313).
Advanced Marfey’s Analysis of Amino Acids. Hydrolysis of han-
tupeptin A (1, 1.0 mg) was achieved in 1.0 mL of 6 N HCl placed in
a sealed reaction vial at 110 °C for 18 h. Trace HCl was removed
under an N2 stream, and the resulting hydrolysate redissolved in 0.6
mL of H2O. The aqueous hydrolysate was divided into two equal
portions. To one portion were added a 1% solution of 1-fluoro-2,4-
dinitrophenyl-5-L-alaninamide (L-FDAA, Marfey’s reagent, 100 µL)
in acetone and 1 M NaHCO3 (25 µL), and the mixture was heated at
40 °C for 45 min. To the second portion was added a racemic mixture
of a 1% solution of 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-
FDAA, 50 µL) in acetone, a 1% solution of 1-fluoro-2,4-dinitrophenyl-
5-D-alaninamide (D-FDAA, 50 µL), and 1 M NaHCO3 (25 µL), and
the mixture was heated at 40 °C for 45 min. Both reaction mixtures
were cooled to RT and quenched by addition of 2 N HCl (25 µL),
dried, and dissolved in MeCN (500 µL). The aliquots were subjected
to reversed-phase LCMS (Agilent 1100 series) according to the
advanced Marfey method10,11 [column: Phenomenex, Luna, 150 × 2.0
mm, 5 µm, 100 Å; mobile phase, MeCN in 0.1% (v/v) aqueous
HCOOH; flow rate, 0.20 mL/min] using a linear gradient (10-50%
MeCN over 60 min). An Agilent 1100 series MSD spectrometer was

32 Journal of Natural Products, 2009, Vol. 72, No. 1
Tripathi et al.
used for detection in API-ES (positive mode). The retention times and
ESIMS product ions (tR in min, m/z [M + H]+) of the L-FDAA
monoderivatized amino acids in the hydrolysate of the first portion were
observed to be Pro (34.9, 366.8), Val (43.0, 368.8), and N-Me-Val (48.4,
382.9), while the reaction with racemic DL-FDAA in the second portion
gave rise to two peaks for each corresponding amino acid moiety. The
retention times and ESIMS product ions (tR1/tR2, min, m/z [M + H]+)
were observed to be Pro (34.9/37.2, 366.8), Val (43.0/49.9, 368.8),
and N-Me-Val (48.4/52.0, 382.8). Peaks eluted with longer tR could be
attributed to the D-FDAA derivative of the amino acids. Consequently,
the absolute configuration of the moieties in the hydrolysate of 1 was
confirmed as L-Pro, L-Val, and N-Me-L-Val. The advanced Marfey
method indicated an N-Me-L-Ile unit in compound 1. However, this
was not conclusive since the N-Me-L-allo-Ile standard was not used.
The N-Me-L-Ile unit in 1 was subsequently confirmed on the basis of
extensive chiral HPLC analyses as well as chemical manipulation using
the advanced Marfey method (see below).
Methanolysis of Hantupeptin A (1). A solution of hantupeptin A
(1, 7.5 mg) in 5% methanolic KOH (0.75 mL) was stirred for 24 h at
RT. The reaction mixture was diluted with ether (15 mL), and the
organic layer was washed with brine, dried over Na2SO4, and
subsequently dried under N2. Purification of 2 (1.5 mg) was ac-
complished by C18 SEP PAK [MeOH/H2O (1:1), MeOH/H2O (3:2),
MeOH/H2O (7:3)].
rate, 0.7 mL/min; detection at 218 nm]. Phenyllactic acid eluted at tR
) 19.5 min, corresponding to the retention time of an authentic sample
of L-3-phenyllactic acid and therefore indicating an S-configuration [tR
of D-3-phenyllactic acid ) 14.7 min].
Cell Viability Assays. Cells were plated in 96-well plates (MOLT-
4, 40 000 cells; MCF-7, 40 000 cells) and were treated 24 h later with
various concentrations of hantupeptin A (1) and solvent control (10%
DMSO). After 24 h of incubation, MTT was added to all wells followed
by lysis buffer. Plates were incubated for another 24 h, and cell viability
was measured by observing absorbance at 570 nm on a plate reader.
Acknowledgment. We acknowledge the SIBiol RTF and NIE AcRF
(RI 8/05 TLT) for financial support. The authors would like to thank
Dr. C. L. Sai (NSSE, NIE) for the use of the Shimadzu Preparative
HPLC system as well as P. Gread (University of Waikato), A. L. Ang
(NSSE, NIE), S. G. Y. Lee (NSSE, NIE), E. J. K. Low (NSSE, NIE),
and C. C. Teo (NSSE, NIE) for technical assistance. In addition, we
would like to thank three anonymous reviewers for their useful
comments.
Supporting Information Available: 1D and 2D NMR data and MS
fragmentation pattern of 1. This material is available free of charge
via the Internet at http://pubs.acs.org.
References and Notes
r-Methoxy-r-trifluoromethyl-r-phenylacetic Acid (MTPA) Es-
ters of 2. Compound 2 obtained from the methanolysis of 1 (see
preceding section) was divided into two equal portions (0.75 mg each),
and to each sample was added 0.75 mL of pyridine. To one portion
was added 6.0 mg of R-MTPACl and to the other 6.0 mg of S-MTPACl,
the reaction was carried out for 10 h at RT, and the solvent was
evaporated under N2. The corresponding esters, 3a and 3b, were
subjected to NMR analysis.
(1) Tan, L. T. Phytochemistry 2007, 68, 954–979.
(2) Gerwick, W. H.; Tan, L. T.; Sitachitta, N. In The Alkaloids; Cordell,
G. A., Ed.; Academic Press: San Diego, 2001; Vol. 57, pp 75-184.
(3) Ramaswamy, A. V.; Flatt, P. M.; Edwards, D. J.; Simmons, T. L.;
Han, B.; Gerwick, W. H. In Frontiers in Marine Biotechnology;
Proksch, P., Muller, W. E. G., Eds.; Horizon Bioscience, 2006; pp
175-224.
(4) (a) Harrigan, G. G.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.;
Park, P. U.; Biggs, J.; Paul, V. J.; Mooberry, S. L.; Corbett, T. H.;
Valeriote, F. A. J. Nat. Prod. 1998, 61, 1221–1225. (b) Luesch, H.;
Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat. Prod. 1999, 62,
1702–1706.
1
3a: H NMR (CDCl3) δ 3.135 (H-2), 4.910 (H-3), 1.652 (H1-4),
1.653 (H-5), 1.511 (H2-6), 1.942 (H-8), 1.260 (H-9), 6.800 (NH), 4.754
(H-11), 2.061 (H-12), 0.980 (H-13), 0.950 (H-14), 2.965 (s, N-Me).
1
3b: H NMR (CDCl3) δ 3.102 (H-2), 4.922 (H-3), 1.673 (H1-4),
(5) (a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Mooberry,
S. L. J. Nat. Prod. 2000, 63, 611–615. (b) Milligan, K. E.; Marquez,
B. L.; Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2000, 63,
1440–1443. (c) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.
J. Nat. Prod. 2000, 63, 1437–1439.
1.682 (H-5), 1.538 (H2-6), 1.953 (H-8), 1.220 (H-9), 6.750 (NH), 4.744
(H-11), 2.040 (H-12), 0.95 (H-13), 0.920 (H-14), 2.940 (s, N-Me).
Racemization of the N-Methyl-L-isoleucine Standard. Approxi-
mately 0.4 mg of the N-Me-L-Ile standard was dissolved in H2O (100
µL), followed by the addition of triethylamine (40 µL) and acetic
anhydride (40 µL). The mixture was heated at 60 °C for 1 h and
evaporated to dryness in a stream of N2. The residue was subsequently
dissolved in 6 N HCl (200 µL), heated at 110 °C for 12 h, and then
evaporated to dryness in a stream of N2.
Advanced Marfey’s Analysis of the Racemized N-Methyl-L-
isoleucine Standard. After racemization of the N-Me-L-Ile standard
[containing both N-Me-L-Ile and N-Me-D-allo-Ile], the advanced Marfey
method (using both L-Marfey’s and D-Marfey’s reagents) was used to
determine the retention times of the four stereoisomers of N-Me-L-Ile
using LCMS [column, Phenomenex, Luna, 150 × 2.0 mm, 5 µ, 100
Å; mobile phase, MeCN in 0.1% (v/v) aqueous HCOOH; flow rate,
0.20 mL/min using a linear gradient (10-50% MeCN over 70 min)].
The retention times and ESIMS product ions (tR in min) of the L/D-
FDAA-derivatized amino acids were as follows: N-Me-L-Ile (56.1),
N-Me-D-allo-Ile (60.3), N-Me-D-Ile ()D-FDAA-derivatized N-Me-L-
Ile, 59.9), and N-Me-L-allo-Ile ()D-FDAA-derivatized N-Me-D-allo-
Ile, 56.6). The tR of the N-Me-Ile unit in the hydrolysate of 1 was similar
to the L-FDAA-derivatized N-Me-L-Ile standard at 56.1 min, and this
was confirmed with coelution of both molecules.
(6) Jaspars, M.; Linda, A. L. Curr. Opp. Drug DiscoVery DeV. 1998, 1,
77–84.
(7) (a) Medina, R. A.; Goeger, D. E.; Hills, P.; Mooberry, S. L.; Huang,
N.; Romero, L. I.; Ortega-Barria, E.; Gerwick, W. H.; McPhail, K. L.
J. Am. Chem. Soc. 2008, 20, 6324–6325. (b) Taori, K.; Paul, V. J.;
Luesch, H. J. Am. Chem. Soc. 2008, 6, 1806–1807. (c) Wrasidlo, W.;
Mielgo, A.; Torres, V. A.; Barbero, S.; Stoletov, K.; Suyama, T. L.;
Klemke, R. L.; Gerwick, W. H.; Carson, D. A.; Stupack, D. G. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 2313–2318.
(8) Tan, L. T.; Chang, Y. Y.; Tripathi, A. Phytochemistry 2008, 69, 2067–
2069.
(9) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(10) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K.-I.
Anal. Chem. 1997, 69, 3346–3352.
(11) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K.-I. Anal. Chem.
1997, 69, 5146–5151.
(12) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 11, 4092–4096.
(13) Ferna´ndez, R.; Rodry´guez, J.; Quinoa´, E.; Riguera, R.; Munoz, L.;
Ferna´ndez-Sua´rez, M.; Debitus, C. J. Am. Chem. Soc. 1996, 118,
11635–11643.
(14) Nakao, Y.; Yoshida, W. Y.; Szabo, C. M.; Baker, B. J.; Scheuer, P. J.
J. Org. Chem. 1998, 63, 3272–3280.
Absolute Configuration of the 3-Phenyllactic Acid Unit in 1.
Hantupeptin A (1, 1.0 mg) was hydrolyzed in 6 N HCl at 110 °C for
12 h. The hydrolysate was concentrated to dryness and analyzed by
chiral HPLC [column, Chiralpak MA(+) (0.46 × 5 cm), Daicel
Chemical Industries Ltd., solvent, 2 mM CuSO4/MeCN (85:15); flow
(15) Bunyajetpong, S.; Yoshida, W. Y.; Sitachitta, N.; Kaya, K. J. Nat.
Prod. 2006, 11, 1539–1542.
(16) Smith, J. A.; Pease, L. G. CRC Crit. ReV. Biochem. 1980, 8, 315–399.
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